Tissue-specific Nitroreductase/Metronidazole (NTR/Mtz) cell ablation system. A: Mechanism of NTR/Mtz cell ablation: a cell expressing the bacterial NTR (NTR⁺; in cyan blue) when exposed to the prodrug Mtz (in green) converts the latter into a cytotoxic agent (in red), which causes DNA damage and death of the NTR⁺ cell. B,D: Constructs used to generate the Tg(cmlc2:CFP-NTR)^s890 and Tg(l-fabp:CFP-NTR)^s891 lines contain the cmlc2:CFP-NTR and l-fabp:CFP-NTR sequences flanked by tol2 sequences. C: The Tg(ins:CFP-NTR)^s892 sequence is flanked by I-Sce sites ("i"). E-G: Brightfield combined with fluorescence imaging of 4, 3, and 6 days postfertilization (dpf) larvae, respectively, showing stable expression of Cyan Fluorescent Protein-NTR (CFP-NTR; reported by CFP fluorescence) in cardiomyocytes (autofluorescence in the yolk, E), Insulin-producing pancreatic β-cells (F) and hepatocytes (G).

Tissue-specific damage to the heart upon exposure of Tg(cmlc2:CFP-NTR)^s890 larvae to Metronidazole (Mtz). A,B: Brightfield images of a control Tg(cmlc2:GFP) larva (A) and Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larva (B) exposed to 10 mM Mtz (in 0.2% dimethyl sulfoxide [DMSO]) at 48 hours postfertilization (hpf) for 24 hr. A′,B′: Fluorescence images of the larvae in A,B, showing Tg(cmlc2:GFP) expression (green). C-J: Confocal images of the heart taken after Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae were exposed to 0.2% DMSO alone (C,E,H), or 10 mM Mtz in 0.2% DMSO (D,F,G,I,J), from 58 to 76 hpf. C,D: Ventricles of DMSO (C) and Mtz-treated (D) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae stained for F-actin (red), Tg(cmlc2:GFP) expression shown in green. E-G: Heart sections of DMSO (E) and Mtz-treated (F,G) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae immunostained for activated Caspase-3 (blue) and stained with rhodamine phalloidin (red), Tg(cmlc2:GFP) in green. G: Same larva as in F at a higher magnification and thicker optical section. H-J: Heart sections of DMSO (H) and Mtz-treated (I,J) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae processed for terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection (red), Tg(cmlc2:GFP) in green. Scale bars = 20 μm. Exposure of 48 hpf embryos to 10 mM Mtz for 24 hr did not affect control larvae (A,A′), but resulted in a severe functional cardiac phenotype in the CFP-NTR⁺ larvae (B,B′): collapsed atrium and ventricle, shown by Tg(cmlc2:GFP) expression (B′), and consequent failure in blood circulation with blood pooling (arrow in B). Confocal imaging of the CFP-NTR/Mtz-mediated damaged ventricle shows a significant loss of actin filaments as well as changes in the overall heart ventricle shape and dimensions (D), compared with ventricles of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae exposed to DMSO only (C). Cell death was detected in hearts of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae exposed to 10 mM Mtz - both by immunostaining for activated Caspase-3 (F,G) and TUNEL assay, in the ventricle (I) as well as in the atrium (J) - whereas in control Tg(cmlc2:CFP-NTR)^s890 larvae treated with DMSO alone, no Caspase-3 expression (E) or TUNEL (H) was detected. TUNEL and Caspase-3 expression was detected exclusively in Tg(cmlc2:GFP)-expressing cells (F,G,I,J), indicating that cell death occurred specifically in cardiomyocytes and without affecting the neighboring CFP-NTR-negative cells.

Functional heart tissue recovery after Nitroreductase/Metronidazole (NTR/Mtz) -mediated cell ablation. A-D: Brightfield images of a control Tg(cmlc2:GFP) larva (A,B) and Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larva (C,D) exposed to 10 mM Mtz at 48 hours postfertilization (hpf), after 24 hr incubation in Mtz (t1; A,C) and after 96 hr washing-out in Mtz-free medium (t2; B,D). E: Brightfield image of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larva incubated in 10 mM Mtz continuously from 48 to 168 hpf (t2). A′,B′,C′,D′,E′: Fluorescence images showing Tg(cmlc2:GFP) expression (green) in the larvae above (A′ corresponding to A, B′ to B, C′ to C, D′ to D, and E′ to E). Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae whose heart tissue was severely damaged as a result of exposure to Mtz for 24 hr (C, C′) were able to replace damaged tissue by functional tissue upon removal of the drug: after 96 hr in Mtz-free medium, the larvae showed a morphologically wild-type heart (D′) and no blood pooling (D). Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae continuously exposed to Mtz for 120 hr showed enhanced cardiac and body phenotype defects: curved body, large yolk edema, small eyes, enlarged eye pockets (E), and collapsed, nonfunctional heart chambers of significantly reduced size (E′).

Ablation and recovery of pancreatic β-cells in zebrafish larvae. A,B: Confocal projections of 96 hours postfertilization (hpf) Tg(ins:CFP-NTR)^s892 principle islets from larvae treated for 12 hr with dimethyl sulfoxide (DMSO) control (A) or Metronidazole (Mtz; B) and immunostained for CFP (cyan) and activated Caspase-3 (red). A: Caspase-3 staining is not observed in Cyan Fluorescent Protein-Nitroreductase-positive (CFP-NTR⁺) β-cells in DMSO-treated larvae. B: Strong staining for activated Caspase-3 and rounded β-cell morphology are seen in β-cells of Mtz-treated larvae (for clarity, A′ and B′ show only the Caspase-3 red channel fluorescence). C-H: Isolated pairs of Tg(ins:CFP-NTR)^s892 larvae monitored during the course of DMSO control (C,E,G) or Mtz prodrug treatment (D,F,H) by combined brightfield/ fluorescence microscopy (scale bars = 250 μm). Inset images are confocal projections of the pancreatic islet of similarly treated sibling larvae, which have been immunostained for Insulin (green) and Glucagon (red; scale bars = 50 μm). C,D: Untreated 84 hpf Tg(ins:CFP-NTR)^s892 larvae show strong CFP-NTR expression in the pancreatic islet (arrows). E,F: 110 hpf Tg(ins:CFP-NTR)^s892 larvae, 26 hr after addition of 0.1% DMSO vehicle or 5 mM Mtz in 0.1% DMSO. E: Islet development appears unaffected after exposure to DMSO (arrows); a centralized "core" of Insulin⁺ β-cells is enveloped by a shell of Glucagon⁺ α-cells (inset). F: After exposure to Mtz, the majority of β-cells have been ablated (asterisks). The surrounding α-cells appear to collapse into the resulting void, forming a more condensed mass; remaining β-cells are clustered nearest the extrapancreatic duct (inset, arrowhead). G,H: 145 hpf Tg(ins:CFP-NTR)^s892 larvae, 35 hr after washout of DMSO or Mtz. G: Pancreatic islet size and structure appears unaffected in DMSO (26 hr) /wash (35 hr) larvae (arrows and inset). H: Significant recovery of β-cell mass is evident in Mtz (26 hr) per wash (35 hr) larvae. Insulin⁺ β-cells repopulating the islet surround a centralized mass of Glucagon⁺ α-cells: an architecture inverse to the original one.

Hepatocyte damage mediated by the Nitroreductase/Metronidazole (NTR/Mtz) system. A-D: Confocal images of hepatic tissue of 7 days postfertilization (dpf) wild-type (A,B) and Tg(l-fabp:CFP-NTR)^s891 (C,D) larvae exposed to dimethyl sulfoxide (DMSO) alone (A,C), or to10 mM Mtz (B,D) from 4 to 7 dpf, immunostained for Prox1 (blue) and F-actin (red). E,E′,F,F′: Confocal images of liver tissue of 5 dpf wild-type (E,E′) and Tg(l-fabp:CFP-NTR)^s891 (F,F′) larvae exposed to 10 mM Mtz from 2 to 5 dpf and immunostained for Prox 1 (blue) and Alcam (red). E′,F′: Prox1 channel of images E and F, respectively. Scale bars = 10 μm. G-I: Confocal images of transverse sections of a 7 dpf wild-type control larva (G) and of a Tg(l-fabp:CFP-NTR)^s891 larva exposed to 10 mM Mtz from 4 to 7 dpf (H; magnified liver section, I, I′) immunostained for Prox1 (green), Alcam (blue), and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL, red). I′: Single channel confocal image of I showing TUNEL staining. A-D: Hepatocytes expressing CFP-NTR under the control of the l-fabp promoter, after treatment with Mtz from 4-7 dpf (D) show a different morphology from the respective controls: CFP-NTR^-DMSO (A), CFP-NTR^-Mtz (B), and CFP-NTR⁺ DMSO (C). In the damaged livers, hepatocytes show an abnormal shape and a significant increase in the size of the nuclei, marked by Prox1 expression (F,F′). However, bile duct cells (surrounded by Alcam expression) do not appear affected (in their nuclear shape or intensity of Prox1 expression; F,F′) compared with the CFP-NTR^- control (E,E′). Immunostaining and TUNEL detection assay on transverse sections of Mtz-treated Tg(l-fabp:CFP-NTR)^s891 larvae (H) vs. Mtz-treated CFP-NTR^- larvae (G) reveal specific cell death in the liver (H,I,I′), whereas no, or almost no, cell death could be detected in nonhepatic tissue or in control larvae (G).

Tissue-specific damage to the heart upon exposure of Tg(cmlc2:CFP-NTR)^s890 larvae to Mtz. Merged images and single channels of heart sections of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae treated with DMSO- (A-A’’’- magnification of Fig. 2E) and Mtz- (B-B’’’ - same as Fig. 2G) immunostained for activated Caspase-3 in blue (A’, B’) and stained with rhodamine phalloidin in red (A’’, B’’), Tg(cmlc2:GFP) in green (A’’’, B’’’). Activated Caspase-3 expression (B’) was detected exclusively in Tg(cmlc2:GFP) expressing cells (B’’’) indicating that cell death occured specifically in cardiomyocytes and without affecting the neighboring CFP-NTR- cells.